A polypeptide targeting VEGFR2, a fluorescent probe and application thereof

Abstract

This invention provides a VEGFR2-targeting peptide, a fluorescent probe, and their applications. The amino acid sequence of the VEGFR2-targeting peptide is shown in SEQ ID No: 1, or the VEGFR2-targeting peptide is a derivative peptide obtained by modifying the amino acid sequence shown in SEQ ID No: 1. The VEGFR2-targeting peptide is coupled with ICG-NHS to obtain the fluorescent probe ICG-VTP. This fluorescent probe has a stable structure, a long plasma half-life, and can specifically recognize and bind to the VEGFR2 receptor. It can be effectively enriched at tumor sites and is suitable for precise fluorescence imaging of tumors, especially ovarian cancer. It helps to accurately identify cancer tissue boundaries during ovarian cancer resection surgery and can diagnose tumors, especially ovarian cancer.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine, and in particular to a polypeptide targeting VEGFR2, a fluorescent probe and application. BACKGROUND

[0002] Ovarian cancer (OC) is one of the most common malignant tumors in the female reproductive system, and it faces many challenges in early diagnosis, including lack of effective screening methods, atypical clinical symptoms, etc. In addition, during the clinical operation, it is still difficult to accurately define the tumor boundary, which directly affects the formulation of intraoperative decision-making and the accuracy of pathological sampling. In view of the close relationship between the overexpression of specific receptors in the tumor occurrence process and disease progression, it is of great clinical significance and application value to develop a new type of molecular probe that can be used for real-time navigation and precise treatment of ovarian cancer during operation.

[0003] Vascular endothelial growth factor receptor 2 (VEGFR2) is a core molecule that regulates tumor angiogenesis. In solid tumors, VEGFR2 significantly enhances endothelial cell proliferation, migration and vascular permeability by mediating the VEGF signaling pathway, supporting tumor growth and metastasis. In the occurrence and development of ovarian cancer, VEGFR2 promotes tumor growth, invasion and metastasis by mediating tumor angiogenesis and lymphangiogenesis. VEGFR2 is highly expressed on the surface of endothelial cells in many tumors, making it an ideal molecular target.

[0004] In the field of tumor targeted diagnosis and treatment, imaging probes or anti-tumor drugs constructed using ligands such as antibodies, affibodies, and polypeptides that can specifically recognize tumor-associated antigens can significantly improve the detection sensitivity of tumor tissue. In the development of targeting molecules, antibodies have long been dominant, but their large molecular weight, high immunogenicity, and high preparation cost have limited their promotion in some clinical application scenarios. In contrast, polypeptides not only have high affinity and high specificity, but also have low immunogenicity, simple molecular structure, small size, and rapid clearance in the body. In addition, polypeptides have better flexibility in chemical modification and coupling, which is suitable for modification strategies that antibodies cannot achieve. Various polypeptides have been confirmed to be high-specificity targeting ligands for recognizing and binding tumor-related biomarkers, and have shown important value in the design of targeted probes and drugs. Therefore, it is of great significance to develop a polypeptide with high specificity for VEGFR2 receptors.

[0005] Near-infrared two region (NIR-II) fluorescent dye indocyanine green (ICG) exhibits significant advantages of strong tissue penetration and low tissue autofluorescence background in in vivo imaging. In addition, the dye has excellent biocompatibility and low toxicity characteristics. The emission spectrum of ICG can be extended to the near-infrared two region, which helps to further improve the quality of in vivo imaging. However, ICG itself does not have tumor targeting and cannot specifically recognize cancer cells. Due to the characteristics of its molecular structure being easy to modify, it can provide a structural basis for constructing targeted fluorescent probes, thereby having potential application prospects in precise tumor imaging. SUMMARY

[0006] The purpose of the present application is to provide a polypeptide targeting VEGFR2, a fluorescent probe and a preparation method and application thereof. The fluorescent probe is prepared by coupling a polypeptide specifically targeting VEGFR2 receptor with near-infrared fluorescent dye indocyanine green (ICG). The probe can specifically recognize tumor cells and lesions with high expression of VEGFR2 under in vivo and in vitro conditions, and is suitable for precise fluorescent imaging and surgical navigation of tumors.

[0007] The technical solutions of the present application are as follows.

[0008] In a first aspect, the present application provides a polypeptide targeting VEGFR2, wherein the amino acid sequence of the polypeptide targeting VEGFR2 is shown in SEQ ID No: 1, or the polypeptide targeting VEGFR2 is a derivative polypeptide obtained by modifying the amino acid sequence shown in SEQ ID No: 1.

[0009] The modifier of the modification treatment is selected from polyethylene glycol and a protecting group.

[0010] The protecting group is selected from at least one of Boc, Fmoc, Adpoc, Iboc, Poc, Z (OMe), Tmz, Cbz and Ddz.

[0011] Preferably, the amino acid sequence of the polypeptide targeting VEGFR2 is HTMYYHHYQHHLSSSDICLPRWGCLWED, as shown in SEQ ID No: 1, and the structural formula is as shown in Figure 1 .

[0012] In a second aspect, the present application provides a fluorescent probe targeting VEGFR2, wherein the preparation raw material of the fluorescent probe targeting VEGFR2 comprises the above-mentioned polypeptide targeting VEGFR2.

[0013] Preferably, the structural formula of the fluorescent probe targeting VEGFR2 is

[0014] ;

[0015] Wherein, R1 is the polypeptide targeting VEGFR2 as described above.

[0016] The structure of the fluorescent probe targeting VEGFR2 (fluorescent probe ICG-VTP) is shown in Figure 4

[0017] In a third aspect, the present application provides a preparation method of the polypeptide targeting VEGFR2, comprising: taking Asp as an initial amino acid, connecting the initial amino acid protected by Fmoc to a solid phase carrier, and removing the Fmoc protection; according to the amino acid sequence of the polypeptide targeting VEGFR2, repeating the following steps: coupling the amino acid protected by Fmoc, removing the Fmoc protection group, obtaining the polypeptide targeting VEGFR2 connected to the solid phase carrier, cleaving, and purifying to obtain the polypeptide targeting VEGFR2.

[0018] In a fourth aspect, the present application provides a preparation method of the fluorescent probe targeting VEGFR2, comprising: dissolving ICG-NHS in an organic solvent, adding the polypeptide targeting VEGFR2, N, N-diisopropylethylamine, and carrying out a light-avoiding reaction, purifying to obtain the fluorescent probe targeting VEGFR2.

[0019] Preferably, the organic solvent is DMSO, and the time for controlling the light-avoiding reaction is 10-14 h.

[0020] In a fifth aspect, the present application provides an application of the polypeptide targeting VEGFR2, the fluorescent probe targeting VEGFR2, or the fluorescent probe targeting VEGFR2 prepared by the preparation method in the preparation of a biological imaging reagent.

[0021] Preferably, the biological imaging reagent is a biological imaging reagent for tumor diagnosis.

[0022] Preferably, the biological imaging reagent for tumor diagnosis is a biological imaging reagent for tumor diagnosis with high expression of VEGFR2.

[0023] Preferably, the biological imaging reagent for tumor diagnosis is a biological imaging reagent for ovarian cancer diagnosis.

[0024] In a sixth aspect, the present application provides an application of the polypeptide targeting VEGFR2, the fluorescent probe targeting VEGFR2, or the fluorescent probe targeting VEGFR2 prepared by the preparation method in the preparation of a resection contour indicating reagent for tumor resection surgery.

[0025] Preferably, the resection contour indicating reagent for tumor resection surgery is a resection contour indicating reagent for ovarian cancer resection surgery.

[0026] The present application has the following beneficial effects: ​

[0027] 1. This invention provides a peptide targeting VEGFR2. Using the VEGFR2-targeting peptide K237 as the targeting moiety, this invention combines it with an albumin-binding peptide (ABP) to construct a new VEGFR2-targeting peptide. The obtained VEGFR2-targeting peptide is then coupled with ICG-NHS to construct a novel targeting fluorescent probe, ICG-VTP. This fluorescent probe has a stable structure, a long plasma half-life, and can specifically recognize and bind to the VEGFR2 receptor, achieving targeted localization of ovarian cancer tissue. This invention also provides a method for synthesizing the above-mentioned VEGFR2-targeting peptide, which has low synthesis costs.

[0028] 2. This invention provides a fluorescent probe targeting VEGFR2, which includes the aforementioned peptide targeting VEGFR2. It can be effectively enriched and retained for a long time at the tumor site, making it suitable for precise fluorescence imaging of tumors, especially ovarian cancer. It has a high signal-to-background ratio, which helps to accurately identify the boundaries of cancerous tissue in ovarian cancer resection surgery, thereby improving the accuracy of tumor resection and preserving normal tissue to the greatest extent.

[0029] 3. This invention also provides the application of the above-mentioned VEGFR2-targeting peptides and fluorescent probes in the preparation of bioimaging reagents. Attached Figure Description

[0030] Figure 1 The structural formula of the polypeptide VTP;

[0031] Figure 2 The results are from the mass spectrometry analysis of the peptide VTP.

[0032] Figure 3 The SPR detection results for peptide VTP;

[0033] Figure 4 The structural formula of the fluorescent probe ICG-VTP;

[0034] Figure 5 This is a mass spectrometry analysis of the fluorescent probe ICG-VTP;

[0035] Figure 6 The spectrum characterization of the fluorescent probe ICG-VTP is shown below; where (A) is the absorption spectrum of the fluorescent probe ICG-VTP; and (B) is the emission spectrum of the fluorescent probe ICG-VTP.

[0036] Figure 7 Structural formula and mass spectrometry analysis of peptide K237;

[0037] Figure 8 Fluorescent probe and mass spectrometry analysis for the fluorescent probe ICG-K237;

[0038] Figure 9 The images show a comparison of the imaging effects of fluorescent probes ICG-VTP and ICG-K237. (A) shows the fluorescence imaging of the femoral vein of Balb / c mice at different time points after injection (5 min, 10 min, 20 min, 30 min, 40 min, 50 min, 60 min, 90 min); (B) shows a comparison of the blood clearance rates of fluorescent probes ICG-VTP and ICG-K237.

[0039] Figure 10 NIR-II fluorescence imaging results of tumor-bearing mice after tail vein injection of fluorescent probe ICG-VTP; (A) shows NIR-II fluorescence images at different time points in the experimental group (injected with fluorescent probe ICG-VTP) and the blocking group (injected with fluorescent probe ICG-VTP and peptide VTP); (B) shows the changes in the tumor / background ratio over time in the experimental group (injected with fluorescent probe ICG-VTP) and the blocking group (injected with fluorescent probe ICG-VTP and peptide VTP).

[0040] Figure 11 For tumor resection surgery mediated by fluorescent probe ICG-VTP imaging;

[0041] Figure 12 This shows the distribution of the fluorescent probe ICG-VTP in vivo. Detailed Implementation

[0042] The present invention will be further described below with reference to specific embodiments. However, these embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Unless otherwise specified, specific conditions in the following embodiments are performed under conventional conditions or conditions recommended by the manufacturer. Unless otherwise specified, the methods used are conventional methods known in the art, and the consumables and reagents used are commercially available. Unless otherwise stated, the technical and scientific terms used herein have the same meaning as those familiar to those skilled in the art. Furthermore, any methods or materials similar to or equivalent to those described herein may also be applied to the present invention.

[0043] Balb / c nude mice and Balb / c mice (female, 6-8 weeks old) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.; human ovarian cancer SKOV3 cells were obtained from the Chinese Academy of Sciences; acetonitrile, dimethyl sulfoxide (DMSO), N,N-diisopropylethylamine (DIEA), trifluoroacetic acid (TFA), dichloromethane (DCM), and N,N-dimethylformamide (DMF) were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; ICG-NHS was purchased from Xi'an Ruixi Biotechnology Co., Ltd.; O-benzotriazole-tetramethylurea hexafluorophosphate (HBTU), triisopropylsilane (TIS), and piperidine (PIP) were purchased from Sinopharm Chemical Reagent Co., Ltd.; resin and amino acids were purchased from Shanghai Jier Biochemical Technology Co., Ltd.; DMEM culture medium was purchased from Thermo Fisher Scientific (China) Co., Ltd.

[0044] Example 1: Preparation of the fluorescent probe ICG-VTP

[0045] The amino acid sequence of the polypeptide VTP is HTMYYHHYQHHLSSSDICLPRWGCLWED, as shown in SEQ ID No: 1.

[0046] Preparation of peptide VTP (a peptide targeting VEGFR2) by solid-phase synthesis

[0047] (1) On the peptide synthesizer, set the corresponding program according to the target peptide sequence. Weigh 1.26 g of MBHA resin and place it in a reaction flask. Add dichloromethane (DCM) to swell for 30 min. Then remove DCM and wash the resin three times with N,N-dimethylformamide (DMF). After each wash, dry the resin thoroughly. Then add the amino acid Fmoc-Asp(OtBu)-OH (3 mmol) protected by the first fluorene methoxycarbonyl (Fmoc) group at the C-terminus, the activating reagent O-benzotriazole-tetramethylurea hexafluorophosphonate (HBTU) (4.5 mmol) in DMF solution, and the base catalyst N,N-diisopropylethylamine (DIEA) (9 mmol) in DMF solution. React for 45 min and wash with DMF, DCM and DMF to obtain Fmoc-Asp(OtBu)-resin.

[0048] (2) Removal of Fmoc: A DMF solution containing 20% ​​(v / v) piperidine was added to the reaction flask to remove the Fmoc protecting group at the amino terminus of the amino acid. After the reaction was completed, the resin was washed with DMF, DCM and DMF respectively to obtain Asp(OtBu)-resin.

[0049] (3) Coupling: The second amino acid at the C-terminus of the target sequence was fixed onto the Asp(OtBu)-resin obtained in step (2). The Asp(OtBu)-resin obtained in step (2), Fmoc-protected amino acid Fmoc-Glu(OtBu)-OH (3 mmol), DMF solution of activating reagent O-benzotriazole-tetramethylurea hexafluorophosphonate (HBTU) (4.5 mmol), and DMF solution of base catalyst N,N-diisopropylethylamine (DIEA) (4.5 mmol) were added to the reaction flask. The reaction was carried out for 45 min. The mixture was washed with DMF, DCM, and DMF to obtain Fmoc-Glu(OtBu)-Asp(OtBu)-resin. Then, Fmoc is removed using the method described in step (2) to obtain Glu(OtBu)-Asp(OtBu)-resin. The process of coupling amino acids and removing Fmoc is then repeated as described above. The Glu(OtBu)-Asp(OtBu)-resin is sequentially coupled with Fmoc-Trp(Boc)-OH, Fmoc-Leu-OH, Fmoc-L-Cys(Trt)-OH, Fmoc-Gly-OH, Fmoc-Trp(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Pro-OH, Fmoc-Leu-OH, Fmoc-L-Cys(Trt)-OH, and Fmoc-Gly-OH. The following peptides, c-Ile-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Leu-OH, Fmoc-His(Trt)-OH, Fmoc-His(Trt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-His(Trt)-OH, Fmoc-His(Trt)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Met-OH, Fmoc-Thr(tBu)-OH, and Fmoc-His(Trt)-OH, are coupled together, and Fmoc is removed. After washing and vacuum drying, the peptide VTP containing resin is obtained.

[0050] (4) Lysis: Based on the amount of resin-containing peptide VTP, prepare a lysis buffer of the appropriate volume at a ratio of 10:1 (v / w) of lysis buffer to resin-containing peptide VTP. The lysis buffer consists of 94% trifluoroacetic acid, 2% triisopropylsilane, 2% ethylenedithiol, and 2% water by volume. Add the lysis buffer to the resin-containing peptide VTP obtained in step (3); stir for 3-4 hours at room temperature; then, separate the solid and liquid, and collect the filtrate; add pre-cooled anhydrous diethyl ether to the filtrate, and centrifuge to obtain a solid product; wash the obtained solid three times with pre-cooled anhydrous diethyl ether, collect the precipitate, and vacuum dry to obtain crude peptide VTP product.

[0051] (5) Purification: The crude peptide VTP obtained in step (4) was purified by high performance liquid chromatography (HPLC). The HPLC column used was a reversed-phase C18 column (XBridge BEH C18 0BD Prep Column). Acetonitrile and water were used as the mobile phase for gradient elution. The mobile phase A was acetonitrile (containing 0.1% TFA), and the mobile phase B was water (containing 0.1% TFA). The gradient elution time was 15 min, the flow rate was 15 mL / min, and the gradient elution conditions were: 33% A + 67% B at 2 min; 80% A + 20% B at 12.5 min; and 20% A + 80% B at 15 min. The detection wavelength was 250 nm. The molecular weight of the target component was determined to be 3514.93 by mass spectrometry, and the corresponding eluent was collected. Subsequently, the acetonitrile was removed by concentration and lyophilized to obtain a white powdery target peptide, which is peptide VTP. The structural formula of peptide VTP is as follows. Figure 1 As shown, the mass spectrometry analysis results of the peptide VTP are as follows: Figure 2 As shown, the surface plasmon resonance (SPR) detection results of the peptide VTP are as follows: Figure 3 As shown.

[0052] Preparation of fluorescent probe ICG-VTP (a fluorescent probe targeting VEGFR2)

[0053] (1) Dissolve 1 mg ICG-NHS in 600 μL LDMSO, add 15.3 mg peptide VTP and 3.3 μL N,N-diisopropylethylamine (DIEA), and react at 37 °C in the dark for 12 h to obtain a reaction solution containing the fluorescent probe ICG-VTP.

[0054] (2) Purification of the reaction solution containing the fluorescent probe ICG-VTP: The reaction solution containing the fluorescent probe ICG-VTP was diluted with methanol and purified by high performance liquid chromatography (HPLC) under the following conditions: the chromatographic column was a reversed-phase C18 column (XBridge BEH C18 0BD Prep Column), gradient elution was used, the mobile phase A was acetonitrile (containing 0.1% TFA), the mobile phase B was water (containing 0.1% TFA), the gradient elution time was 24.5 min, the flow rate was 8 mL / min, and the gradient elution conditions were: 10% A + 90% B at 2 min; 50% A + 50% B at 16 min; 95% A + 5% B at 18 min; 10% A + 90% B at 24.5 min, and the detection wavelength was 250 nm. The final green product was confirmed as the expected product ICG-VTP by HPLC and mass spectrometry analysis.

[0055] The structural formula of ICG-VTP is as follows: Figure 4 As shown. The mass spectrometry analysis results of ICG-VTP are as follows. Figure 5 As shown. The absorption and emission spectra are as follows. Figure 6 As shown.

[0056] Example 2: Preparation of fluorescent probe ICG-K237

[0057] The amino acid sequence of polypeptide K237 is HTMYYHHYQHHL, as shown in SEQ ID No: 2.

[0058] The solid-phase synthesis method for preparing peptide K237 includes the following steps:

[0059] (1) On the peptide synthesizer, set the corresponding program according to the target peptide sequence. Weigh 1.26 g of MBHA resin and place it in a reaction flask. Add dichloromethane (DCM) to swell for 30 min, then remove DCM and wash the resin three times with N,N-dimethylformamide (DMF). After each wash, dry the resin thoroughly. Add a DMF solution of Fmoc-protected amino acid Fmoc-Leu-OH (3 mmol), activating reagent O-benzotriazole-tetramethylurea hexafluorophosphonate (HBTU) (4.5 mmol), and base catalyst N,N-diisopropylethylamine (DIEA) (9 mmol). React for 45 min and wash with DMF, DCM, and DMF to obtain Fmoc-Leu-resin.

[0060] (2) Removal of Fmoc: A DMF solution containing 20% ​​(v / v) piperidine was added to the reaction flask to remove the Fmoc protecting group at the amino terminus of the amino acid. After the reaction was completed, the mixture was washed with DMF, DCM and DMF respectively to obtain Leu-resin.

[0061] (3) Repeat the process of coupling amino acids and removing Fmoc protecting groups in steps (1) and (2) to couple Leu-resin sequentially with Fmoc-His(Trt)-OH, Fmoc-His(Trt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-His(Trt)-OH, Fmoc-His(Trt)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Met-OH, Fmoc-Thr(tBu)-OH, and Fmoc-His(Trt)-OH, and remove Fmoc, wash, and vacuum dry to obtain the resin-containing polypeptide K237.

[0062] (4) Lysis: Based on the amount of resin-containing polypeptide K237, prepare a lysis buffer at a ratio of 10:1 (v / w) of lysis buffer to crude resin-containing polypeptide K237. The lysis buffer consists of 94% trifluoroacetic acid, 2% triisopropylsilane, 2% ethylenedithiol, and 2% water by volume. Add the lysis buffer to the crude resin-containing polypeptide K237 obtained in step (3); stir for 3-4 hours at room temperature; then separate the solid and liquid, and collect the filtrate; add pre-cooled anhydrous diethyl ether to the filtrate, and obtain the solid product by centrifugation; wash the obtained solid three times with pre-cooled anhydrous diethyl ether, collect the precipitate, and dry it under vacuum to obtain the crude polypeptide K237 product.

[0063] (5) Purification: The crude product of polypeptide K237 was purified. The purification method of the crude product of polypeptide K237 was the same as that of the crude product of polypeptide VTP in Example 1. The target polypeptide, which is white powder, was obtained.

[0064] The structural formula and mass spectrometry results of peptide K237 are as follows: Figure 7 As shown.

[0065] The preparation method of the fluorescent probe ICG-K237 includes the following steps:

[0066] (1) Dissolve 1 mg ICG-NHS in 600 μL LDMSO, add 6.03 mg peptide K237 and 3.3 μL DIEPA, and react at 37 °C in the dark for 12 h to obtain a reaction solution containing the fluorescent probe ICG-K237.

[0067] (2) The method for purifying the reaction solution containing the fluorescent probe ICG-K237 is the same as the method for purifying the reaction solution containing the fluorescent probe ICG-VTP in Example 1. The final green product was confirmed by HPLC and mass spectrometry analysis to be the expected product, the fluorescent probe ICG-K237.

[0068] The structural formula and mass spectrometry results of the fluorescent probe ICG-K237 are as follows: Figure 8 As shown.

[0069] Example 3: Testing of fluorescent probes ICG-VTP and ICG-K237

[0070] 1) Blood half-life assay of fluorescent probes ICG-VTP and ICG-K237 in Balb / c mice

[0071] To determine the plasma half-life of two fluorescent probes (ICG-VTP and ICG-K237), Balb / c mice were injected with equal doses (20 nmol) of each probe via the tail vein. Fluorescence imaging was performed on the femoral vein region of the mice's abdomen, and the clearance rate of the probes in vivo was calculated based on changes in fluorescence signal. Results are as follows: Figure 9 As shown, Figure 9 (A) shows the fluorescence imaging of the femoral vein of Balb / c mice at different time points (5 min, 10 min, 20 min, 30 min, 40 min, 50 min, 60 min, and 90 min) after injection in different experimental groups (fluorescent probe ICG-VTP and ICG-K237 groups). Figure 9 (B) in the figure is a comparison of the blood clearance rates of fluorescent probes ICG-VTP and ICG-K237. Figure 9 The normalized fluorescence intensity in (B) was calculated as 100% of the vascular fluorescence intensity at 5 min. Figure 9 As shown in Figure (A), both fluorescent probes could clearly image the femoral vein at 5 minutes post-injection; however, the femoral vein signal of fluorescent probe ICG-K237 became blurred at 40 minutes and almost undetectable at 60 minutes, indicating a faster plasma clearance rate. In contrast, fluorescent probe ICG-VTP could still clearly display the femoral vein structure at 60 minutes and maintained good visibility until signal blurring occurred at 90 minutes. Figure 9 The quantitative analysis results in (B) further confirmed that the blood half-life of the fluorescent probe ICG-VTP was significantly longer than that of the fluorescent probe ICG-K237. The specific plasma half-lives of the fluorescent probes ICG-VTP and ICG-K237 were calculated using the following formulas: the plasma half-life of the fluorescent probe ICG-K237 was 10.84 min; the plasma half-life of the fluorescent probe ICG-VTP was 30.24 min.

[0072] The specific formula is as follows: T (1 / 2) = 0.693 / K (Formula 1)

[0073] K=[ln(Int1)–ln(Int2)] / (t2-t1) (Formula 2)

[0074] T (1 / 2) The value represents the plasma half-life in minutes; K = elimination rate constant; Int1 is the fluorescence intensity at t1; Int2 is the fluorescence intensity at t2; in this test, t1 was 5 minutes and t2 was 60 minutes.

[0075] 2) Optical imaging of the fluorescent probe ICG-VTP in SKOV3 human ovarian cancer-bearing mice

[0076] SKOV3 tumor model establishment: Each 5×10 6 One SKOV3 cell was resuspended in 100 μL of DMEM basal medium, and each mouse was injected with 100 μL of this culture subcutaneously in the right shoulder. The transplanted tumor was allowed to grow to approximately 100-200 mm in size. 3 At that time, in vivo imaging experiments were performed on tumor-bearing mice.

[0077] Experimental group: The fluorescent probe ICG-VTP was prepared into a 125 μM solution with physiological saline. 200 μL of the fluorescent probe ICG-VTP solution was injected into mice via the tail vein. Optical signals were collected at time points of 0.5, 1, 2, 4, 6, 8, 10, 12, 24 and 48 hours after injection to observe the distribution of the fluorescent probe ICG-VTP in mice and its enrichment in the tumor area.

[0078] Blocking group: The fluorescent probe ICG-VTP was prepared into a 125 μM solution with physiological saline, and peptide VTP was added to the solution to make the molar concentration of peptide VTP 30 times that of the fluorescent probe ICG-VTP, i.e., 3.75 mM, to obtain a mixed solution containing peptide VTP and fluorescent probe ICG-VTP. 200 μL of the mixed solution was injected into mice via the tail vein. Optical signals were collected at time points of 0.5, 1, 2, 4, 6, 8, 10, 12, 24 and 48 hours after injection to observe the distribution of fluorescent probe ICG-VTP in mice and its enrichment in the tumor area.

[0079] The NIR-II fluorescence imaging results at different time points for the experimental group (injected with fluorescent probe ICG-VTP) and the blocking group (injected with fluorescent probe ICG-VTP and peptide VTP) are as follows: Figure 10As shown in Figure (A), SKOV3 represents the experimental group, and Block represents the blocking group. The results show that in the experimental group, starting 4 hours after injection, the fluorescent probe ICG-VTP showed significant aggregation in the tumor area, and the tumor margin gradually became clearer; 48 hours after injection, the fluorescent probe ICG-VTP remained in the tumor area. Optical imaging results at various time points clearly show that after blocking with peptide VTP, no obvious fluorescent signal was observed at the tumor site, indicating that peptide VTP successfully competed for the binding site. The ability of the tumor site to take up the probe is due to the specific binding of the probe to VEGFR2. The changes in the tumor / background ratio (signal-to-background ratio) over time for the experimental group (injected with fluorescent probe ICG-VTP) and the blocking group (injected with both fluorescent probe ICG-VTP and peptide VTP) are shown in Figure (A). Figure 10 As shown in (B), SKOV3 represents the experimental group, and Block represents the blocking group. The experimental results show that the tumor / background signal ratio (signal-to-background ratio) reached its highest level in the experimental group 8 hours after injection. This demonstrates that the fluorescent probe ICG-VTP, used as a contrast agent, exhibits high-quality imaging, particularly in significantly improving the tumor-to-background signal ratio.

[0080] 3) Tumor resection and in vivo distribution mediated by the fluorescent probe ICG-VTP

[0081] like Figure 11 As shown, under the guidance of NIR-II fluorescence imaging using the fluorescent probe ICG-VTP, precise tumor identification and complete resection were achieved by differentiating the fluorescence signals of tumor tissue, surrounding skin, and muscle. While thoroughly removing the tumor tissue, the muscle tissue with weaker fluorescence signals was effectively preserved, thus achieving complete tumor removal while ensuring surgical safety. Mice were processed 48 hours after tumor resection imaging, and major organs and tissues were collected to evaluate the biodistribution of the fluorescent probe ICG-VTP in vivo. The results are as follows: Figure 12 As shown.

[0082] The above description is merely a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any modifications, equivalent substitutions, and improvements made by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the invention.

Claims

1. A polypeptide targeting VEGFR2, characterized in that, The amino acid sequence of the peptide targeting VEGFR2 is shown in SEQ ID No:

1.

2. A fluorescent probe targeting VEGFR2, characterized in that, The raw materials for preparing the fluorescent probe targeting VEGFR2 include the peptide targeting VEGFR2 as described in claim 1.

3. The fluorescent probe targeting VEGFR2 according to claim 2, characterized in that, The structural formula of the fluorescent probe targeting VEGFR2 is as follows: ； Wherein, R1 is the peptide targeting VEGFR2 as described in claim 1.

4. A method for preparing the peptide targeting VEGFR2 as described in claim 1, characterized in that, include: Using Asp as the initial amino acid, the initial amino acid protected by Fmoc is linked to a solid support, and the Fmoc protection is removed. The following steps are repeated according to the amino acid sequence of the peptide targeting VEGFR2: coupling the Fmoc-protected amino acid, removing the Fmoc protecting group, to obtain the peptide targeting VEGFR2 linked to the solid support, cleaving, and purifying to obtain the peptide targeting VEGFR2.

5. A method for preparing a fluorescent probe targeting VEGFR2 as described in claim 2 or 3, characterized in that, include: ICG-NHS was dissolved in an organic solvent, and the peptide targeting VEGFR2 as described in claim 1 and N,N-diisopropylethylamine were added. The mixture was reacted in the dark and purified to obtain the fluorescent probe targeting VEGFR2.

6. The method for preparing a fluorescent probe targeting VEGFR2 according to claim 5, characterized in that, The organic solvent is DMSO, and the reaction time in the dark is controlled to be 10-14 hours.

7. The application of the peptide targeting VEGFR2 according to claim 1, the fluorescent probe targeting VEGFR2 according to claim 2 or 3, and the fluorescent probe targeting VEGFR2 obtained by the preparation method according to claim 5 or 6 in the preparation of bioimaging reagents for ovarian cancer diagnosis.

8. The use of the peptide targeting VEGFR2 according to claim 1, the fluorescent probe targeting VEGFR2 according to claim 2 or 3, and the fluorescent probe targeting VEGFR2 obtained by the preparation method according to claim 5 or 6 in the preparation of a resection contour indicator reagent for ovarian cancer resection surgery.

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